Inactivating bacteria with electric pulses and antibiotics

ABSTRACT

Provided is a method of reducing a number of viable microbes, including contacting microbes with an antibiotic compound and applying pulses of electricity having a duration of between about 50 nanoseconds and about 900 nanoseconds. The pulses of electricity may have an intensity between about 20 kV/cm and about 40 kV/cm. The pulses of electricity may be applied at a frequency of between about 0.1 Hz and about 10 Hz. The microbes may be a gram-negative or a gram-positive strain of bacteria and the antibiotic may be applied at a concentration for a duration, wherein applying the antibiotic to the strain at the concentration for the duration does not reduce a viable number of bacteria of the strain as much, or at all, when the pulses of electricity are not also applied.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/991,284, filed Aug. 12, 2020, which is a continuation ofInternational Application No. PCT/US2019/017761, filed on Feb. 13, 2019,and published on Aug. 22, 2019, and claims benefit of priority from U.S.Provisional Patent Application No. 62/630,219, filed Feb. 13, 2018, theentire contents of which are hereby incorporated by reference in theirentirety.

GOVERNMENT RIGHTS STATEMENT

This invention was made with Government support under grant numberNRC-HQ-84-14-G-0048 awarded by the U.S. Nuclear Regulatory Commission.The Government has certain rights in the invention.

FIELD OF THE INVENTION

The subject matter disclosed herein relates to inhibiting bacteria byapplying antibiotics thereto in combination with the application ofvoltage pulses. More specifically, this disclosure relates to applyingelectric pulses of nanosecond duration in combination with theapplication of antibiotics, the application of both of which incombination may result in an enhanced inhibition or prevention ofbacterial growth, proliferation, or survival.

BACKGROUND OF THE INVENTION

Antimicrobial resistance (AMR) is a major challenge and growing globalhealth crisis, with many scientists and public health professionalssounding the alarm on a “post-antibiotic” era. While the Center forDisease Control (CDC) and WHO have issued statements and guidelinesconcerning antibiotic use and antibiotic resistant microbes andinitiated tracking protocols at medical facilities, this does not fullyaddress the underlying causes for resistance development and emergence.The overuse of antibiotics in agriculture and medicine is a key driverof antibiotic resistance and high production animal farming methodspromote the spread of disease in overcrowded spaces. Farmers often applyineffective levels of antibiotics that improve animal growth rates.However, this allows the bacteria in that environment to more easilydevelop resistance. In medical settings, antibiotics are oftenprescribed for non-bacterial infections. Additionally, doctorsfrequently apply broad spectrum antibiotics rather than identifying thecausative organism and applying a targeted treatment due to the lack ofrapid, low-cost testing methods. Patients often stop taking medicationupon becoming asymptomatic; however, the surviving microorganisms canacquire resistance to subsequent treatments with the same antibiotic.

The increased obsolescence of antibiotics, combined with the slow paceof new antibiotic development, especially in combatingcarbapenem-resistance in gram negative bacteria, such as K pneumoniae,and fluoroquinolone resistance in P aeruginosa has forced medicalfacilities to more frequently treat gram negative infections with olderdrugs, such as colistin, drugs considered treatments of last resort.Currently, gram-positive infections, such as Methicillin-resistantStaphylococcus aureus (MRSA) comprise a significant portion ofclinically relevant bacteria, and numerous novel antimicrobial drugshave been licensed to target them in the past decade. While the list ofessential medicines includes many drugs for combating gram-positiveinfections, several of these drugs, such as linezolid and vancomycin,have no clinically significant effect on most gram-negative bacteria.

The scientific and medical communities are currently exploring methodsto combat this problem, including the development of new monitoring anddiagnostic tools to improve data collection, in vitro systems to mimicevolutionary environments to determine the time between antibioticsusceptibility and resistance, and methods to interfere with existingresistance mechanisms while simultaneously developing new antibiotics.The latter method is one of escalation, since any use of antibiotics inhumans or animals will breed resistance in the surviving microbes, whichcan acquire transmittable genetic material. Subsequent generations ofthese surviving microbes require higher doses of that antibiotic,ultimately necessitating the development of a new class of antibiotics.While new techniques, such as detecting antimicrobial compounds as theyare produced in their natural environment in the soil instead of a Petridish using electronic chips, will facilitate antibiotic development.However, the traditional life cycle of these drug development methodsand clinical use will inevitably result in stronger, more resistantsuperbugs.

Thus, there remains a need to improve the antibacterial regimen withlower doses of antibiotics to prevent development of resistance and toincrease spectrum of bacteria susceptible to treatment with a givenantibacterial regimen. The present disclosure is directed to overcomingthese and other deficiencies in the art.

SUMMARY OF THE INVENTION

In one aspect, provided is a method of reducing a number of viablemicrobes, including contacting microbes with an antibiotic compound andapplying pulses of electricity having a duration of between about 50nanoseconds and about 900 nanoseconds. In an embodiment, the pulses ofelectricity have an intensity and the intensity is between about 20kV/cm and about 40 kV/cm. For example, the intensity may be about 20 kV/cm, or may be about 30 kV/cm, or may be about 40 kV/cm. In anotherembodiment, the pulses of electricity are applied at a frequency ofbetween about 0.1 Hz and about 10 Hz.

In yet another embodiment, the microbes are a strain of bacteria and theantibiotic is applied at a concentration for a duration, whereinapplying the antibiotic to the strain at the concentration for theduration does not reduce a viable number of bacteria of the strain whenthe pulses of electricity are not also applied to the strain. In anexample, the strain is a gram-negative strain. In another example, thestrain is a gram-positive strain.

In another embodiment, the microbes are a strain of bacteria and theantibiotic is applied at a concentration for a duration, wherein thereducing is greater when the antibiotic is applied to the strain at theconcentration for the duration when the pulses of electricity areapplied to the strain than when the antibiotic is applied to the strainat the concentration for the duration when the pulses of electricity arenot applied to the strain. In an example, the strain is a gram-negativestrain. In another example, the strain is a gram-positive strain.

In a further embodiment, the microbes are a strain of bacteria, whereinapplying the pulses of electricity at the intensity to the strain doesnot reduce a viable number of bacteria of the strain when the strain isnot also contacted with an antibiotic. In an example, the strain is agram-negative strain. In another example, the strain is a gram-positivestrain.

In still another embodiment, the antibiotic is selected from at leastone of an aminoglycoside antibiotic, an ansamycin antiobiotic, abeta-lactam antibiotic, a glycopeptide antibiotic, a lincosamideantibiotic, a lipopeptide antibiotic, a macrolide antibiotic, amonobactam antibiotic, a nitrofuran antibiotic, an oxazolidinoneantibiotic, a quinolone antibiotic, a fluoroquinolone antibiotic, asulfonamide antibiotic, a tetracycline antibiotic, pexiganan, fusidicacid, mupirocin, and any combination of at least two of the foregoing.In still a further embodiment, the antibiotic is selected from at leastone of tobramycin, streptomycin, rifampicin, vancomycin, clindamycin,daptomycin, erythromycin, linezolid, penicillin, minocycline, pexiganan,fusidic acid, mupirocin, bacitracin, neomycin, polymixin B,metronidazole, silver, zinc, copper, and any combination of at least twoof the foregoing.

In yet a further embodiment, the microbe is Staphylococcus aureus,Staphylococcus epidermidis, Escherichia coli, Acenitobacter baumanii,Klebsiella pneumoniae, or Pseudomonas aeruginosa. In another embodiment,the microbe is vancomycin-resistant Staphylococcus aureus,methicillin-resistant Staphylococcus aureus, a strain ofmultidrug-resistant Pseudomonas aeruginosa, or a strain ofmultidrug-resistant Escherichia coli.

In a further embodiment, contacting further includes administering theantibiotic to a human, and applying further includes applying theelectric pulses to the human. In an example, administering is selectedfrom administering topically and administering systemically.

In another aspect, provided is a method of reducing a number of viablemicrobes, including contacting microbes with an antibiotic compound andapplying pulses of electricity having a duration of between about 50nanoseconds and about 900 nanoseconds, wherein the pulses of electricityhave an intensity and the intensity is between about 20 kV/cm and about40 kV/ cm. For example, the intensity may be about 20 kV/ cm, or may beabout 30 kV/ cm, or may be about 40 kV/ cm. In another embodiment, thepulses of electricity are applied at a frequency of between about 0.1 Hzand about 10 Hz.

In yet another embodiment, the microbes are a strain of bacteria and theantibiotic is applied at a concentration for a duration, whereinapplying the antibiotic to the strain at the concentration for theduration does not reduce a viable number of bacteria of the strain whenthe pulses of electricity are not also applied to the strain. In anexample, the strain is a gram-negative strain. In another example, thestrain is a gram-positive strain.

In still another embodiment, the microbes are a strain of bacteria andthe antibiotic is applied at a concentration for a duration, wherein thereducing is greater when the antibiotic is applied to the strain at theconcentration for the duration when the pulses of electricity areapplied to the strain than when the antibiotic is applied to the strainat the concentration for the duration when the pulses of electricity arenot applied to the strain. In an example, the strain is a gram-negativestrain. In another example, the strain is a gram-positive strain.

In a further embodiment, the microbes are a strain of bacteria, whereinapplying the pulses of electricity at the intensity to the strain doesnot reduce a viable number of bacteria of the strain when the strain isnot also contacted with an antibiotic. In an example, the strain is agram-negative strain. In another example, the strain is a gram-positivestrain.

In still another embodiment, the antibiotic is selected from at leastone of an aminoglycoside antibiotic, an ansamycin antiobiotic, abeta-lactam antibiotic, a glycopeptide antibiotic, a lincosamideantibiotic, a lipopeptide antibiotic, a macrolide antibiotic, amonobactam antibiotic, a nitrofuran antibiotic, an oxazolidinoneantibiotic, a quinolone antibiotic, a fluoroquinolone antibiotic, asulfonamide antibiotic, a tetracycline antibiotic, pexiganan, fusidicacid, mupirocin, and any combination of at least two of the foregoing.In still a further embodiment, the antibiotic is selected from at leastone of tobramycin, streptomycin, rifampicin, vancomycin, clindamycin,daptomycin, erythromycin, linezolid, penicillin, minocycline, pexiganan,fusidic acid, mupirocin, bacitracin, neomycin, polymixin B,metronidazole, silver, zinc, copper, and any combination of at least twoof the foregoing.

In yet a further embodiment, the microbe is Staphylococcus aureus,Staphylococcus epidermidis, Escherichia coli, Acenitobacter baumanii,Klebsiella pneumoniae, or Pseudomonas aeruginosa. In another embodiment,the microbe is vancomycin-resistant Staphylococcus aureus,methicillin-resistant Staphylococcus aureus, a strain ofmultidrug-resistant Pseudomonas aeruginosa, or a strain ofmultidrug-resistant Escherichia coli.

In a further embodiment, contacting further includes administering theantibiotic to a human, and applying further includes applying theelectric pulses to the human. In an example, administering is selectedfrom administering topically and administering systemically.

In yet another aspect, provided is a method of reducing a number ofviable microbes, including contacting microbes with an antibioticcompound and applying pulses of electricity having a duration of betweenabout 50 nanoseconds and about 900 nanoseconds. In an embodiment, thepulses of electricity have an intensity and the intensity is betweenabout 20 kV/cm and about 40 kV/cm. In an example, the intensity is about20 kV/cm. In another example, the intensity is about 30 kV/cm. In yetanother example, the intensity is about 40 kV/cm. In another embodiment,the pulses of electricity are applied at a frequency of between about0.1 Hz and about 10 Hz.

In an example, the antibiotic includes an aminoglycan. In anotherexample, the antibiotic includes tobramycin. In yet another example, theantibiotic includes an ansamycin. In still another example, theantibiotic comprises rifampicin.

In a further example, the microbe includes a strain of Stapphylococcusaureus. In yet a further example, the microbe includes a strain ofEscherichia coli. In still a further example, the antibiotic includes aglycopeptide.

In another example, the antibiotic includes vancomycin. In yet anotherexample, the antibiotic includes an oxazolidinone. In still anotherexample, the antibiotic includes linezolid. In a further example, theantibiotic includes an ansamycin. In yet a further example, theantibiotic includes rifampicin. In still a further example, theantibiotic includes mupirocin. In an example, the antibiotic includes amacrolide. In another example, the antibiotic includes erythromycin. Inyet another example, the antibiotic includes fusidic acid.

In another example, the microbe includes a multidrug-resistant strain ofEscherichia coli. In yet another example, the microbe includesKlebsiella pneumoniae. In still another example, the microbe includes amultidrug-resistant strain of Pseudomonas aeruginosa.

In a further example, the microbes are a strain of bacteria, whereinapplying the pulses of electricity at the intensity to the strain doesnot reduce a viable number of bacteria of the strain when the strain isnot also contacted with an antibiotic. In an example, the strain is agram-negative strain. In another example, the strain is a gram-positivestrain.

In still a further example, contacting further includes administeringthe antibiotic to a human, and applying further includes applying theelectric pulses to the human. In yet another example, administering isselected from administering topically and administering systemically.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentdisclosure will become better understood when the following detaileddescription is read with reference to the accompanying drawings,wherein:

FIG. 1 shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentrations of the tobramycin synergistically reducedan amount of viable S. aureus.

FIG. 2 shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotic tobramycinsynergistically reduced an amount of viable E. coli with the effectstronger for higher electric fields.

FIG. 3 shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotic rifampicinsynergistically reduced an amount of viable S. aureus.

FIG. 4 shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotic rifampicinsynergistically reduced an amount of viable E. coli.

FIG. 5A shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotic tobramycinsynergistically reduced an amount of viable S. aureus.

FIG. 5B shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotic tobramycinsynergistically reduced an amount of viable E. coli.

FIG. 6 shows effects of combining trains of 300 ns electric pulses withthe same energy, but different electric fields and number of pulses,with different concentration of the antibiotics tobramycin andrifampicin (same concentration of each drug) synergistically reduced anamount of viable methicillin-resistant S. aureus.

FIG. 7 shows synergistically reduced amount of viablemethicillin-resistant S. aureus under 300 ns electric pulses combinedwith administration of different antibiotics.

FIG. 8A and FIG. 8B show reduced amount of viable methicillin-resistantS. aureus following 300 ns electric pulses (EPs) (445 at 20 kV/cm or 222at 30 kV/cm) and/or various concentrations of antibiotics (from left toright, Lin=linezolid, FA=fusidic acid, Erth=erythromycin, Mup=mupirocin,Rif=rifampicin, and Van=vancomycin). FIG. 8A Combining EPs at eitherelectric field enhances MRSA inactivation compared drug alone with the30 kV/cm EPs inducing greater inactivation for both concentrations ofall drugs. FIG. 8B. Synergy induced by combining EPs with antibiotics(0-log indicates no synergy). Combining the 20 kV/cm EP with 2 μg/mLrifampicin induced a statistically significant synergistic inactivationcompared to the same concentration of other antibiotics studied.

FIG. 9A and FIG. 9B show reduced amount of viable K. pneumoniaefollowing treatment with 300 ns electric pulses (EPs) (445 at 20 kV/cmand 222 at 30 kV/cm) and/or various concentrations of antibiotics (fromleft to right, Lin=linezolid, Mup=mupirocin, Rif=rifampicin, andVan=vancomycin). FIG. 9A. Applying the 30 kV/cm EPs induces over a 3-logreduction in K. pneumoniae and combining EPs with 2 μg/mL or 20 μg/mL ofmupirocin, rifampicin, vancomycin results in statistically significantincreases in bacteria inactivation from 4-log to 5-log. FIG. 9B. Synergyinduced by combining EPs with antibiotics (0-log indicates no synergy).Combining 2 μg/mL or 20 μg/mL of mupirocin, rifampicin, or vancomycinwith the 30 kV/cm EPs induced at least approximately 1-log synergy.

FIG. 10A and FIG. 10B show reduced amount of viable E. coli followingtreatment with 300 ns electric pulses (EPs) (445 at 20 kV/cm and 222 at30 kV/cm) and/or various concentrations of antibiotics (from left toright, Lin=linezolid, FA=fusidic acid, Erth=erythromycin, Mup=mupirocin,Rif=rifampicin, and Van=vancomycin). FIG. 10A Applying 20 kV/cm EPsinduces approximately 1-log reduction and combining the 20 kV/cm EPswith either 2 μg/mL or 20 μg/mL of any of the antibiotics inducesapproximately 2-log to 3-log reduction, which is statisticallysignificant for the 2 μg/mL doses. Applying the 30 kV/cm EPs inducesapproximately a 3-log reduction in E. coli and combining these EPs with2 μg/mL or 20 μg/mL of any of the antibiotics induces statisticallysignificant reductions of 3-log to 5-log and 3-log to 6-log,respectively. FIG. 10B Synergy induced by combining EPs with antibiotics(0-log indicates no synergy). Adding 2 μg/mL of any drug except forvancomycin to the 20 kV/cm EPs induced at least 1-log of statisticallysignificant synergy. Adding 20 μg/mL of linezolid and rifampicin inducedapproximately 2-log of statistically significant synergy. Combining 2μg/mL or 20 μg/mL of any of the drugs considered with the 30 kV/cm EPsinduced at least 1-log synergy.

FIG. 11A and FIG. 11B show reduced amount of viable P. aeruginosafollowing treatment with 300 ns electric pulses (EPs) (445 at 20 kV/cmand 222 at 30 kV/cm) and/or various concentrations of antibiotics (fromleft to right, Lin=linezolid, Erth=erythromycin, Mup=mupirocin,Rif=rifampicin, and Van=vancomycin). FIG. 11A Applying 20 kV/cm EPsinduces approximately 1-log reduction and combining the 20 kV/cm EPswith either 2 μg/mL or 20 μg/mL of any of the antibiotics inducesapproximately 2-log to 3-log reduction, which is statisticallysignificant for the 2 μg/mL doses. Applying the 30 kV/cm EPs inducesapproximately a 3-log reduction in E. coli and combining these EPs with2 μg/mL or 20 μg/mL of any of the antibiotics induces statisticallysignificant reductions of 3-log to 5-log and 3-log to 6-log,respectively. FIG. 11B Synergy induced by combining EPs with antibiotics(0-log indicates no synergy). Adding 2 μg/mL of any drug except for Vanto the 20 kV/cm EPs induced at least 1-log of statistically significantsynergy. Adding 20 μg/mL of linezolid and rifampicin inducedapproximately 2-log of statistically significant synergy. Combining 2μg/mL or 20 μg/mL of any of the drugs considered with the 30 kV/cm EPsinduced at least 1-log synergy.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to a method of reducing a number of viablemicrobes. A method may include contacting microbes with an antibioticand applying electric pulses of a duration on the order of nanoseconds.In an aspect, combining an application of electric pulses of a durationon the order of nanoseconds enhances an antibacterial effect of one ormore antibiotics. Thus, in an example, contacting a given strain ofmicrobe with a given concentration of a given antibiotic may not resultin an antimicrobial effect on the microbe in the absence ofcoadministration of electric pulses of nanosecond duration, but whencombined with such electric pulses such antibiotic may have anantibiotic effect.

Electric pulses (EPs) are known to be able to eradicate microorganisms,such as by inducing lysis. Application of EPs has also been employed forsome medical and biological applications, including extending food shelflife, permeabilizing cells to facilitate gene and molecular delivery,permeabilizing tumors to enhance the delivery of chemotherapeutics, anddirectly killing tumors through irreversible electroporation. Suchapplications typically involve direct EP targeting of cell membranesusing electric fields of hundreds of V/cm to a few kV/ cm with durationsfrom microseconds to milliseconds.

Technology development over the past two decades has led to thebiomedical application of nanosecond-duration EPs (NSEPs) (such as EPswith a duration of between about 1 nanosecond and about 1 microsecond)with field strengths ranging from tens of kV/cm to a few hundred kV/cm.These shorter durations enable charging intracellular membranes prior tothe cell membrane, permitting intracellular manipulation with minimalcell membrane impact. Without being limited to any particular theory ofactivity, NSEPs may also permit creating membrane nanopores that enableions and small molecules to enter the cell while prohibiting largermolecules.

Provided in the present disclosure is a method for reducing drug doseand EP energy input which may reduce numbers of viable microbes,including gram-positive and gram-negative bacteria. For example,combining application of electric pulses of nanosecond duration over arelatively brief time frame (e.g., anywhere from about one minute toabout one hour) may enhance an ability of an antibiotic, applied with orwithout one or more additional antibiotics, to reduce viability ofmicrobes to which such treatment is applied. An abbreviated time duringwhich NSEPs may be applied in order to so inhibit viability of microbesin the presence of antibiotics, including enhancing antimicrobialeffectiveness of a concentration of antibiotics applied, mayadvantageously facilitate treatment. For example, NSEPs could be appliedto a subject (e.g., human or animal) over a period of an hour or less(or 30 minutes or less or 20 minutes or less or 15 minutes or less or 10minutes or less of 5 minutes of less or two minutes or less) whileantibiotics are applied (e.g., topically near a site where NSEPs areapplied or systemically such as through any systemic administrationwoute appropriate for a given antibacterial drug). Becauseadministration of NSEPs over a period of more than one hour may be moredifficult to accomplish, a method disclosed herein includingadministration of NSEPs for one hour or less provides significantadvantages over alternatives. In other examples, NSEPs may beadministered across a duration of time in excess of an hour. Forexample, NSEPs may be administered for two, three four, five, six,seven, eight, nine, ten, eleven, twelve, or more hours.

EPs may be administered according to various parameters. Such parametersinclude intensity of EP applied, duration of EP, frequency of EPadministration, number of EPs applied in a train of EPs, number oftrains of EPs applied, and duration of time between trains of EPs.

As to intensity of EPs, as disclosed herein, EPs may be within a rangeof about 20 kV/cm to about 40 kV/cm. In this case, “about” meansintensity of EPs may vary somewhat from these precise values while stillfalling within the intensities as so described. For example, “about” maymean within +/−5% of a value. Intensity may be within such range, orwithin a sub-range thereof. For example, intensity of an EP as disclosedherein may be about 20 kV/cm, about 25 kV/cm, about 30 kV/cm, about 35kV/cm, or about 40 kV/cm Alternatively, EPs may be within a range fromabout 15 kV/cm to about 25 kV/cm, from about 20 kV/cm to about 30 kV/cm,from about 25 kV/cm to about 30 kV/cm, from about 25 kV/cm to about 35kV/cm, from about 30 kV/cm to about 35 kV/cm, from about 30 kV/cm toabout 40 kV/cm, or from about 35 kV/cm to about 40 kV/cm, or within anysubranges within these ranges. In other examples, EPs may have anintensity below about 20 kV/cm. For example, an EP may have an intensityof about 15 kV/cm, or about 10 kV/cm, or about 5 kV/cm, or about 1kV/cm, or any value therebetween. In other examples, an EP may have anintensity above about 40 kV/cm. For example, an EP may have an intensityof about 45 kV/cm, or about 50 kV/cm, or about 55 kV/cm, or about 60kV/cm, or about 70 kV/cm, or about 75 kV/cm, or about 80 kV/cm, or about85 kV/cm, or about 90 kV/cm, or about 100 kV/cm, or any value or rangetherebetween.

As to duration of EP, EPs may be nanosecond-EPs (NSEPs), in that theymay have a duration of between about 1 ns and about 1 microsecond. Inthis case, “about” means duration of EPs may vary somewhat from theseprecise values while still falling within the intensities as sodescribed. For example, “about” may mean within +/−5% of a value. Forexample, NSEPs may be a duration of between about 50 ns and about 900ns, or any value or range therebetween. For example, an NSEP may have aduration of about 50 ns, or about 60 ns, 70 ns, or about 80 ns, or about90 ns, or about 100 ns, or about 110 ns, or about 120 ns, or about 130ns, or about 140 ns, or about 150 ns, or about 160 ns, or about 170 ns,or about 180 ns, or about 190 ns, or about 200 ns, or about 210 ns, orabout 220 ns, or about 230 ns, or about 240 ns, or about 250 ns, orabout 260 ns, or about 270 ns, or about 280 ns, or about 290 ns, orabout 300 ns, or about 310 ns, or about 320 ns, or about 330 ns, orabout 340 ns, or about 350 ns, or about 360 ns, or about 370 ns, orabout 380 ns, or about 390 ns, or about 400 ns, or about 410 ns, orabout 420 ns, or about 430 ns, or about 440 ns, or about 450 ns, orabout 460 ns, or about 470 ns, or about 480 ns, or about 490 ns, orabout 500 ns, or about 510 ns, or about 520 ns, or about 530 ns, orabout 540 ns, or about 550 ns, or about 560 ns, or about 570 ns, orabout 580 ns, or about 590 ns, or about 600 ns, or about 610 ns, orabout 620 ns, or about 630 ns, or about 640 ns, or about 650 ns, orabout 660 ns, or about 670 ns, or about 680 ns, or about 690 ns, orabout 700 ns, or about 710 ns, or about 720 ns, or about 730 ns, orabout 740 ns, or about 750 ns, or about 760 ns, or about 770 ns, orabout 780 ns, or about 790 ns, or about 800 ns, or about 810 ns, orabout 820 ns, or about 830 ns, or about 840 ns, or about 850 ns, orabout 860 ns, or about 870 ns, or about 880 ns, or about 890 ns, orabout 900 ns.

An NSEP may also have a duration within any subrange within about 50 nsto about 900 ns. For example, an NSEP may have a duration of betweenabout 50 ns and 100 ns, about 100 ns and about 150 ns, about 150 ns andabout 200 ns, about 250 ns and about 300 ns, about 300 ns and about 350ns, about 350 ns and about 400 ns, about 400 ns and about 450 ns, about450 ns and about 500 ns, about 500 ns and about 550 ns, about 550 ns andabout 600 ns, about 600 ns and about 650 ns, about 650 ns and about 700ns, about 700 ns and about 750 ns, about 750 ns and about 800 ns, about800 ns and about 850 ns, about 850 ns and about 900 ns, about 100 ns andabout 200 ns, about 200 ns and about 300 ns, about 300 ns and about 400ns, about 400 ns and about 500 ns, about 500 ns and about 600 ns, about600 ns and about 700 ns, about 700 ns and about 800 ns, about 800 ns andabout 900 ns, about 100 ns and about 300 ns, about 300 ns and about 500ns, about 500 ns and about 700 ns, about 700 ns and about 900 ns, about100 ns and about 500 ns, or about 500 ns and about 900 ns.

In some examples, NSEPs may have a duration of less than about 50 ns.For example, an NSEP may have a duration of about 45 ns, about 40 ns,about 35 ns, about 30 ns, about 25 ns, about 20 ns, about 15 ns, about10 ns, about 5 ns, or about 1 ns, or a duration within a rangetherebetween. In other examples, NSEPs may have a duration of longerthan about 900 ns, such as about 910 ns, about 920 ns, about 930 ns,about 940 ns, about 950 ns, about 960 ns, about 970 ns, about 980 ns,about 990 ns, about 995 ns, or about 999 ns. NSEPs have a duration ofless than 1 μs.

In some circumstances, EP of durations of 1 μs or higher maydisadvantageously impair functioning or viability or non-microbialcells. For example, where EPs are administered to a subject such as ahuman subject along with application or administration of anantibacterial or other antimicrobial substance for the purpose ofreducing a number of viable microbes, applying EP with a duration of 1μs or longer may disadvantageous also damage cells and/or tissue of thesubject not just decrease viability of microbes. Thus, application ofNSEPs as disclosed herein explicitly excludes application of EPs with aduration of 1 μs or longer. Surprisingly, as disclosed herein, applyingNSEPs over an abbreviated time frame produces a substantial reduction inmicrobe viability when paired with administration of one or moreantimicrobial agents, including when neither the EP alone orconcentration of antimicrobial agent alone has an effect of reducing anumber of viable microbes or effects a lesser reduction of a number ofviable microbes than does application of either EP or antimicrobialagent alone. Such an effect is found without application of EPs of aduration of 1 μs or longer, thereby enhancing an antimicrobial effectwhich minimizing, reducing, eliminating, or avoiding deleterious effectson cells or tissue of a subject such as a human subject receiving suchtreatment.

In other examples, such as when a severe infection may occur or bepresent, one or more EP of longer then about 1 μs may be administeredeven at the risk of damaging tissue beyond eradicating microbes. Forexample, some damage to tissue may be an acceptable trade-off forreducing a number of viable microbes. Some tissue may be lost duringdebridement procedure that may be provided either before or after theapplication of NSEP and an antimicrobial composition.

EPs may be administered in a series or train of EPs, meaning more thanone NSEP applied in temporally proximate succession. For example,anywhere from 2 to 200 NSEPs may be applied in a train with a frequencyof administration of between about 0.1 Hz and about 10 Hz. In this case,“about” means frequency of EPs may vary somewhat from these precisevalues while still falling within the intensities as so described. Forexample, 2, about 5, about 10, about 12, about 15, about 17, about 20,about 22, about 25, about 27, about 30, about 32, about 35, about 37,about 40, about 42, about 45, about 47, about 50, about 52, about 55,about 57, about 60, about 62, about 65, about 67, about 70, about 72,about 75, about 77, about 80, about 82, about 85, about 87, about 90,about 92, about 95, about 97, about 100, about 102, about 105, about107, about 110, about 112, about 15, about 117, about 120, about 122,about 125, about 127, about 130, about 132, about 135, about 137, about140, about 142, about 145, about 147, about 150, about 152, about 155,about 157, about 160, about 162, about 165, about 167, about 170, about172, about 175, about 177, about 180, about 182, about 185, about 187,about 190, about 192, about 195, about 197, or about 200 EPs may beadministered in a train of between 0.1 Hz to about 10 Hz. In this case,“about” means the number of EPs may be within +/−2 of the numberindicated. Any number of NSEPs or subrange within the foregoingidentified number of NSEPs may also be applied. In an example, betweenabout 15 and about 20, about 10 and about 40, or about 20 and about 100NSEPs may be administered at a frequency of between about 0.1 Hz andabout 10 Hz.

In another example, anywhere from 2 to 1,000 NSEP applied in temporallyproximate succession. For example, anywhere from 2 to 1,000 NSEPs may beapplied in a train with a frequency of administration of between about0.1 Hz and about 10 Hz. In this case, “about” means frequency of EPs mayvary somewhat from these precise values while still falling within theintensities as so described. For example, 2, about 10, about 50, about100, about 150, about 200, about 250, about 300, about 350, about 400,about 450, about 500, about 550, about 600, about 650, about 700, about750, about 800, about 850, about 900, about 950, about 1,000, about1,050, about 1,100, about 1,150, about 1,200, about 1,250, about 1,300,about 1,350, about 1,400, about 1,450, about 1,500, about 1,550, about1,600, about 1,650, about 1,700, about 1,750, about 1,800, about 1,850,about 1,900, about 1,950, about 2,000, about 2,050, about 2,150, about2,100, about 2,150, about 2,200, about 2,250, about 2,300, about 2,350,about 2,400, about 2,450, about 2,500, about 2,550, about 2,600, about2,650, about 2,700, about 2,750, about 2,800, about 2,850, about 2,900,about 2,950, about 2,300, about 2,350, about 2,400, about 2,450, about2,500, about 2,550, about 2,600, about 2,650, about 2,700, about 2,750,about 2,800, about 2,850, about 2,900, about 2,950, or about 3,000 EPsmay be administered in a train of between 0.1 Hz to about 10 Hz. In thiscase, “about” means the number of EPs may be within +/−25 of the numberindicated. In another example, more than about 3,000 NSEPs may beadministered (such as bout 4,000 or about 5,000 or more). Any number ofNSEPs or subrange within the foregoing identified number of NSEPs mayalso be applied. In an example, between about 100 and about 500, about400 and about 800, or about 600 and about 1,000 NSEPs may beadministered at a frequency of between about 0.1 Hz and about 10 Hz.

In some circumstances, EPs numbering in the hundreds or 1,000 or more ata frequency of between about 0.1 Hz and about 10 Hz maydisadvantageously impair functioning or viability or non-microbialcells, particularly at higher intensities (for example, above about 20kV/cm). For example, where EPs are administered to a subject such as ahuman subject along with application or administration of anantibacterial or other antimicrobial substance for the purpose ofreducing a number of viable microbes, applying EPs numbering in thehundreds or 1,000 or more at a frequency of between about 0.1 Hz andabout 10 Hz at higher intensities may disadvantageous also damage cellsand/or tissue of the subject not just decrease viability of microbes.Surprisingly, as disclosed herein, applying NSEPs over an abbreviatedtime frame produces a substantial reduction in microbe viability whenpaired with administration of one or more antimicrobial agents,including when neither the EP alone or concentration of antimicrobialagent alone has an effect of reducing a number of viable microbes oreffects a lesser reduction of a number of viable microbes than doesapplication of either EP or antimicrobial agent alone. Such an effect isfound without application of EPs numbering in the hundreds or 1,000 ormore at a frequency of between about 0.1 Hz and about 10 Hz, therebyenhancing an antimicrobial effect which minimizing, reducing,eliminating, or avoiding deleterious effects on cells or tissue of asubject such as a human subject receiving such treatment.

In other examples, such as when a severe infection may occur or bepresent, one or more EPs numbering in the hundreds or 1,000 or more at afrequency of between about 0.1 Hz and about 10 Hz may be administeredeven at the risk of damaging tissue beyond eradicating microbes. Forexample, some damage to tissue may be an acceptable trade-off forreducing a number of viable microbes. Some tissue may be lost duringdebridement procedure that may be provided either before or after theapplication of NSEP and an antimicrobial composition.

Frequency of administration of NSEPs within a train may be as low asabout 0.1 Hz or as high as about 10 Hz. In an example, frequency isabout 1 Hz. In another example, frequency is between about 0.5 Hz andabout 2 Hz. In another example, frequency may be about 0.5 Hz, about 1.0Hz, about 1.5 Hz, about 2.0 Hz, about 2.5 Hz, about 3.0 Hz, about 3.5Hz, about 4.0 Hz, about 4.5 Hz, about 5.0 Hz, about 5.5 Hz, about 6.0Hz, about 6.5 Hz, about 7.0 Hz, about 7.5 Hz, about 8.0 Hz, about 8.5Hz, about 9.0 Hz, about 9.5 Hz, or about 10 Hz. Trains of pulses mayalso be administered within a range of pulses overlapping thesefrequencies. For example, a train of NSEPs may be administered at afrequency of between about 1 Hz and about 2 Hz, about 1 Hz and about 3Hz, about 1 Hz and about 5 Hz, about 3 Hz and about 5 Hz, about 5 Hz andabout 7.5 Hz, and about 5 Hz and about 10 Hz.

In some examples, NSEPs may have a duration of between about 50 ns andabout 900 ns, may have an intensity of between about 10 kV/cm and about50 kV/cm, and be administered at between about 0.5 Hz and about 1.5 Hzin a train of between 10 to 20 NSEPs administered. However, explicitlyincluded within the present disclosure is different combinations of theforegoing parameters. Any herein disclosed duration of NSEPs having anintensity of between about 10 kV/cm and about 50 kV/cm, and beadministered at between about 0.5 Hz and about 1.5 Hz in a train ofbetween 10 to 20 NSEPs administered, is explicitly included within thepresent disclosure. Any herein disclosed frequency of NSEP of betweenabout 50 ns and about 900 ns may be administered at between about 0.5 Hzand about 1.5 Hz in a train of between 10 to 20 NSEPs administered, isexplicitly included within the present disclosure. Any herein disclosedfrequency of NSEPs administered in a train of NSEPs with intensity ofbetween about 10 kV/cm and about 50 kV/cm disclosed frequency of NSEP ofbetween about 50 ns and about 900 ns may be administered in a train ofbetween 10 to 20 NSEPs administered, is explicitly included within thepresent disclosure. Administration of any number of NSEPs within a trainas disclosed herein having a duration of between about 50 ns and about900 ns, an intensity of between about 10 kV/cm and about 50 kV/cm, andadministered at between about 0.5 Hz and about 1.5 Hz, is explicitlyincluded within the present disclosure.

NSEPs may be generated by a Blumlein circuit, which can be built innumerous configurations using capacitors (based on capacitance/chargestorage devices), including, but not limited to, ceramic basedcapacitors, transmission lines, and other dielectrics (such as water).One can control the EP duration by the Blumlein circuit design either bycontrolling the capacitance or length of the transmission line.Similarly, one can control the pulse shape by modifying the number andnature of the switches to vary the rise- and fall-times, whichinfluences whether the pulse appears square or trapezoidal with respectto time. Increasing the voltage beyond the physical capabilities of thematerials used in the Blumlein circuit can be achieved by using a Marxgenerator, which is a voltage adding device. Typical Marx approachescharge parallel full-bridge switch-capacitor cells at a lower voltage,and through controllable switches, connect in series with a biologicalsample and discharge into the load at a higher voltage as a function ofthe number of series components. The resulting series equivalentcapacitor (Ceq) voltage is discharged into the biological load, which iscalculated as V_(load)≈NV_(C) where N is the number of Marx stages withcapacitors charged to V_(C) and depends on stray system capacitance andinductance. Garner, et al. outline various pulse generator designs thatmay be relevant, including a modular, controllable Marx-based technologydeveloped in collaboration with GE particularly for platelet activation.See A. L. Garner, A. Caiafa, Y. Jiang, S. Klopman, C. Morton, A. S.Torres, A. M. Loveless, and V. B. Neculaes, “Design, Characterizationand Experimental Validation of a Compact, Flexible Pulsed PowerArchitecture for Ex Vivo Platelet Activation,” PLOS ONE, 12(7), e0181214(2017) and A. Caiafa, V. B. Neculaes, A. S. Tones, and A. L. Garner,“Modular Adjustable Pulse Generator,” U.S. Pat. No. 9,238,808 B2 (issued19 Jan. 2016).

In an example, more than one train of NSEPs may be administered. Forexample, two, three, four, five, six, or more trains may beadministered. A duration between trains may be anywhere from between oneminute to about one hour. In some examples, the duration between trainsmay be 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45min, 50 min, 55 min, or 60 min. In an example, administering more thanone train of NSEPs within a duration between trains of 60 min or less,such as 30 min or less or 20 min or less of 15 min or less or 10 min orless or 5 min or less, may advantageously enhance an antimicrobialeffect of a method as disclosed herein (surprisingly given the rapidityof effectiveness) by application of more than one train of NSEPs butwithin an abbreviated time frame for greater ease and improved logisticsof application. Thus, treatment times on the order of minutes inaccordance with the present disclosure may replace what conventionalantimicrobial therapy may require hours or days to attain. In otherexamples, longer durations between trains may be used when desirable oradvantageous or where shorted inter-train intervals are not required ordesired. In an example, trains may be separated by about 15 min or 20min. And of the durations of inter-train intervals as disclosed hereinmay be about or approximately of the durations identified, in that theymay be +/−5% of the duration indicated.

A microbe may be any organism the presence or growth or proliferation ofwhich may be undesirable. For example, a microbe may be a bacteriastrain, such as infectious bacteria. A bacteria strain may begram-positive or gram-negative. Some non-limiting examples of bacteriainclude Staphylococcus aureus, Staphylococcus epidermidis, Escherichiacoli, Acenitobacter baumanii, Klebsiella pneumoniae, and Pseudomonasaeruginosa. Other bacteria related to any of the foregoing species anddifferent strains thereof may also be included in a method as disclosedherein. On the basis of the present disclosure, it would be understoodthat a method of reducing a number of viable microbes hereby providedcould be applied to numerous different microbes in addition to theseexamples. Some specific examples include strains of microbes known to beresistant or show low levels of susceptibility to treatment with knownantibiotics. Examples include vancomycin-resistant Staphylococcusaureus, methicillin-resistant Staphylococcus aureus, a strain ofmultidrug-resistant Pseudomonas aeruginosa, or a strain ofmultidrug-resistant Escherichia coli. Some non-limiting examples ofstrains of bacteria that are known to be resistant or refractory totreatment with one or more antibiotics which are generally effectiveagainst other strains of said bacteria include: carbapenem-resistantEscherichia coli; Klebsiella pneumoniae; gentamicin, streptomycin andsulfonamide resistant Pseudomonas aeruginosa; and methicillin resistantStaphylococcus aureus which in some examples may also be resistant toerythromycin and tetracycline. Such strains show low or nosusceptibility to exposure to various antibiotics that are otherwisebactericidal and/or bacteriostatic to other strains or other bacteria atcomparable concentrations.

In some cases, gram-negative bacteria may be resistant to treatment withan antibiotic that is bacteriostatic or bactericidal as against agram-positive bacteria. Differences in susceptibility betweengram-positive and gram-negative bacteria to a given antibiotic are knownand believed attributable to differences between the bacteria in thethicknesses of peptidylglycan layers in cell walls. Thus, a variety ofantibiotics may be available for having a bactericidal and/orbacteriostatis effect against gram-positive bacteria because ofsusceptibility of or because of cell wall peptidylglycan ingram-positive bacteria to certain antibiotics, whereas differentpeptiglycan cell wall layers in gram-negative may correspond to theirlack of susceptibility to the same antibiotics. Certain gram-negativebacteria in particular are known to show high resistance to treatmentwith certain or broad classes of antibiotics. Surprisingly andadvantageously, as provided herein, disclosed is a method for renderingbacteria susceptible to treatment with a given antibiotic when appliedin the presence of NSEPs as disclosed herein whereas susceptbility isabsent following treatment with the antibiotic in the absence of NSEPs.In other examples, the effectiveness of a dose of an antibiotic may beenhanced by co-administration with NSEPs such that a lower dose orconcentration than would otherwise be effective or necessary becomeseffective when paired with NSEPs.

Antibiotics of any of a number of difference classes or types may beused in accordance with the method disclosed herein. Examples includeaminoglycosides, ansamycins, carbapenems, cephalosporins, antibioticglycopeptides, lincosamides, abitbiotic lipopeptides, macrolides,monobactams, nitrofurans, oxazolidinones, penicillins, quinolones,fluoroquinolones, sulfonamides, tetracyclines, or others. Any antibioticfrom any of these categories may be used in accordance with aspects ofthe present disclosure. Non-limiting specific examples include,tobramycin, streptomycin, rifampicin, vancomycin, clindamycin,daptomycin, erythromycin, linezolid, penicillin, minocycline, pexiganan,fusidic acid, mupirocin, bacitracin, neomycin, polymixin B, andmetronidazole. Other examples include metals or metal ions known to haveantimicrobial or antibacterial effects, such as silver, copper, or zinc.In some examples, combinations of any two or more of the foregoingantibiotics or substances with antibiotic activity may be administeredconcurrently in accordance with an aspect of the present disclosure. Insome examples, any one or more of the foregoing may also be explicitlyexcluded from use in accordance with an aspect of the presentdisclosure.

Other infectious or other microbes may also be included and renderedsusceptible to treatment with NSEPs in combination with application ofan antimicrobial agent (whereas susceptibility was absent upon treatmentwith only the antimicrobial or only the NSEPs, or susceptibility isenhanced upon combined administration of both compared to administrationof either alone). For example, a microbe may be a fungus, such asCandida albicans, Candida auris, or species of Aspergillis. Variousantifungal compounds may also be administered in accordance with anaspect of the present disclosure. Non-limiting examples includeclotrimazole, econazole, miconazole, terbinafine, fluconazole,ketoconazole, and amphotericin, or other compounds known to haveantifungal activities. In some examples, combinations of any two or moreof the foregoing antifungals or substances with antifungal activity maybe administered concurrently in accordance with an aspect of the presentdisclosure. In some examples, any one or more of the foregoingantifungals or substances with antifungal activity may also beexplicitly excluded from use in accordance with an aspect of the presentdisclosure. In some other examples, one or more of the foregoingantibiotics or substances with antibiotic activity may be used incombination with any one or more of the foregoing antifungals orsubstances with antifungal activity in accordance with an aspect of thepresent disclosure.

In an aspect, inhibiting the growth, proliferation, viability,reproduction, infectivity, or number of microbes may be desirable. Asused herein, reducing a number of viable microbes includes any of theforegoing effects on microbe colonies or populations. Included arebacteriostatic and bactericidal effects. An antimicrobial compositionmay be administered with application of NSEPs in accordance with thepresent disclosure with the result of inhibiting the growth,proliferation, viability, reproduction, infectivity, or number ofmicrobes present, each and all of which are included in reducing anumber of viable microbes. A reduction in a number of viable microbesmay result from a strictly bactericidal effect, a bacteriostatic effect,or a combination of the two.

A reduction in a number of viable microbes may be identified by any of anumber of known methods. For example, a treatment may be applied to oneof two otherwise identical samples, then the samples cultured to measuremicrobial growth following said treatment as compared to followingabsence of said treatment. If fewer microbes are present after culturingthe sample to which said treatment had been applied relative to theuntreated sample, the treatment reduced a number of viable microbes. Asample may be any surface, composition, liquid, substance, surface,tissue, or other material to which treatment as disclosed herein may beapplied. In an example, applying NSEPs and an antimicrobial compositionto a subject, such as a human or non-human animal subject, results inless infection (less in severity, less in duration, or both, or absenceof infection) than results under similar circumstances, or than wouldhave resulted, without treatment. For example, application of suchtreatment may slow growth of infectious microbes or otherwise renderthem more susceptible to a subject's immune system. Such examples ofreduced infection are examples of reducing a number of viable microbes.

In some examples, a treatment as disclosed herein may slow or preventproliferation of microbes and thereby hasten a reduction in number ofviable microbes (e.g., increase susceptibility to a subject's immunesystem). In other examples, a treatment as disclosed herein may killmicrobes without immediately eliminating or removing them. Both areexamples of a treatment reducing a number of viable microbes.

In other examples, a reduction in a viable number of microbes might notresult in a reduced duration, degree, or severity of an infection butmay be evinced by culturing a sample and ascertaining an amount ofmicrobial growth supported by such sample (following treatment asopposed to absent treatment). Other measures of a number of viablemicrobes may be used as well, such as quantitative measures of microbialmarkers (antigens, genetic material, etc.) present in a sample, ormicroscopic or other known detection method. In some examples, suchreduction of a number of viable microbes may be evident within about 1hr, about 2 hr, about 3 hr, about 4 hr, about 5 hr, about 6 hr, about 7hr, about 8 hr, about 9 hr, about 10 hr, about 11 hr, about 12 hr, about13 hr, about 14 hr, about 15 hr, about 16 hr, about 17 hr, about 18 hr,about 19 hr, about 20 hr, about 21 hr, about 22 hr, about 23 hr, about24 hr, about 25 hr, about 26 hr, about 27 hr, about 28 hr, about 29, hr,about 30 hr, about 31 hr, about 32 hr, about 33 hr, about 34 hr, about35 hr, about 36 hr, about 37 hr, about 38 hr, about 39 hr, about 40 hr,about 41 hr, about 42 hr, about 43 hr, about 44 hr, about 45 hr, about46 hr, about 47 hr, about 48 hr, about 54 hr, about 60 hr, about 66 hr,about 72 hr, about 78 hr, about 84 hr, about 90 hr, about 96 hr, about4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days,about 7 days, about 10 days, about 14 days, about 17 days, about 21days, or about 28 days after administration of electric pulsescommenced. In this case, “about” means within +/−15% of the durationindicated.

An antimicrobial composition may be applied to a surface, solution, orsubstance, together with application of NSEPs as disclosed, in order toreduce a number of viable microbes on said surface or in such solutionor substance, in accordance with the present disclosure. In an example,an antimicrobial may be applied or administered to a living subject suchas a human and NSEPs applied in accordance with the present disclosure.For example, an acute, chronic, sub-acute, sub-chronic,treatment-refractory, or other microbial infection, such as a bacterialor fungal infection, may be present in a subject such as a humansubject. An antimicrobial composition may be applied or administered tosuch subject and NSEPs applied to reduce a number of viable microbes,such as to eliminate, remove, reduce, ameliorate, or otherwise treatsuch infection. In another example, such infection may be anticipated ora risk of such infection may be present, such as in an immunocompromisedsubject, or in conjunction with surgery or wound or trauma, or known orexpected exposure to an infectious microbe, whereupon an antimicrobialcomposition may be administered with application of NSEPsprophylactically, to prevent development of infection or proliferationof an infectious seed of microbe that may be present or suspected ofbeing present. Such examples are included with reducing an amount ofviable microbes as the term is used herein.

An antimicrobial composition may be administered by any of variousmedically known or accepted or approved means of applying oradministering such antimicrobial composition. Examples include oral,parenteral (including subcutaneous, intradermal, intramuscular,intravenous and intraarticular), rectal and topical (including dermal,buccal, sublingual and intraocular) administration. An antimicrobial maybe formulated as appropriate for such administration, which may betailored to a given purpose, such as in a tablet, capsule, or other formfor oral administration or injectable formulation for injection, or gel,cream, powder, ointment, or other composition for rectal or dermalapplication, etc. In some examples, one or more antimicrobial may beincluded in the surface of a material or an apparatus to be implanted onor within the body of a subject such as a human subject configured orotherwise formulated to have or promote an antimicrobial effect at thesurface of such material or apparatus or to be released therefrom andhave such an antimicrobial effect in tissue in the vicinity of suchmaterial or apparatus.

In accordance with an aspect of the present disclosure, NSEPs may beadministered to such a subject so as to enhance an antimicrobial effectof such antimicrobial composition and reduce a number of viablemicrobes. For example, a subject may receive or may have receivedsystemic treatment with an antimicrobial composition such thatapplication of NSEPs to a part of the subject's body enhances anantimicrobial effect of said antimicrobial composition on microbespresent where NSEPs are applied, reducing a number of viable microbes.In another example, an antimicrobial composition may be topicallyapplied, such as in a cream or ointment or powder or other form, orlocally injected, or present in a material or apparatus implanted or tobe implanted, and NSEPs applied at a site of antimicrobial therebyapplied, to enhance antimicrobial effectiveness or otherwise reduce anumber of viable microbes there. Skilled persons would comprehend thatvarious ways to apply antimicrobial compositions could be used inaccordance with an aspect of the present disclosure.

In an example, an antimicrobial composition or substance may be appliedor present at a concentration, or to achieve a concentration locally,that alone does not have an effect on a number of viable microbes at agiven site. In another example, an antimicrobial composition orsubstance may be applied or present at a concentration, or to achieve aconcentration locally, that alone has only low effect on a number ofviable microbes at a given site. In either example, in accordance withan aspect of the present disclosure, applying NSEPs may increase areduction in a number of viable microbes otherwise resulting fromapplication of the antimicrobial composition in the absence of NSEPs. Anantimicrobial composition may be administered at a concentration that isnot effective at all or only minimally effective at reducing a number ofviable microbes of a given species or strain when applied in the absenceof NSEPs, whereas combining such administration with application ofNSEPs cause an increase in reduction of viable microbes. In anotherexample, NSEPs may be ineffective or only marginally effective or theirown in reducing a number of viable microbes on their own but renderedeffective in the presence of an antimicrobial composition. In bothexamples, neither an antimicrobial on its own or NSEP administration onits own may be effective in reducing a number of viable microbes whereasthe combination of both is. In other examples, application of one or theother, or each, on its own may be somewhat effective but combinedadministration of both antimicrobial composition and NSEP may be moreeffective in reducing a number of viable microbes than either alone.

In another example, a time frame required for effectiveness of anantimicrobial composition in reducing a number of viable microbes may bereduced when administered in combination with application of NSEPs.Conventionally, an antimicrobial composition such as an antibiotic orantifungal may require hours, days, or even weeks to be effective inreducing a number of viable microbes, or to be fully effective inpreventing or eliminating an infection. Surprisingly, as demonstratedherein, an antimicrobial composition may show substantial effectivenessin reducing a number of viable microbes in a short time frame, such aswithin an hour or less, following brief application or applications ofNSEPs. Thus, whereas a given concentration of an antimicrobialcomposition may be effective in reducing a number of viable microbes onits own, combining its administration with application of NSEPs asdisclosed herein may result in reduction of a number of viable microbesfollowing a shorter time span of exposure to the antimicrobialcomposition at that concentration than would otherwise be requiredbefore such an effect of the antimicrobial composition results.

EXAMPLES

The following examples are intended to illustrate particular embodimentsof the present disclosure but are by no means intended to limit thescope thereof.

Synergistic Effects of NSEPS and Antibiotics

Equipment. Used was a capacitor based Blumlein pulse generator with aspark gap switch to produce trapezoidal EPs of 300 ns duration at thepeak with rise and fall times close to 50 ns. Multiple pulses weredelivered at a frequency of 1 Hz.

The samples were treated in a standard electroporation cuvette (DotScientific) whose resistance must match the pulse generator's impedanceto prevent pulse reflection. Utilizing Luria broth, containing 0.5%NaCl, in a cuvette with gap distances of 1 mm and 2 mm yielded the bestelectrical match (no reflections in the measured signal). Used were 2 mmcuvettes to ensure consistency between tests, and the applied voltagewas measured across the cuvette using a LeCroy PPE 20 kV high voltageprobe with a 1000:1 attenuation feeding into a TeleDyne LeCroyWaverunner 6 Zi Oscilloscope capable of measuring signals up to 4 GHz.Assuming a purely parallel plate geometry with minimal fringing gives anelectric field E=V/d, where V is the peak voltage of the applied pulseand d is the gap distance.

Sample Preparation. These experiments assessed Stapphylococcus aureus(gram positive bacterium number 25923) from ATCC and Escherichia coli(gram negative bacterium number 25922) inoculated in luria broth (LBBroth Lennox, powder microbial growth medium, SIGMA-ALDRICH) by taking 8mL of broth in a 50 mL sterile conical tube and incubating in a shakerfor 20 h at 37° C. The sample was then diluted with fresh Luria brothuntil attaining an optical density of 0.25 for 100 μL on aphotospectrometer (Molecular Devices) for a wavelength of 562 nm asmeasured in a 96-well plate. The optical density, which varies withgrowth or tube volume, was determined experimentally and set between1×10⁸ and 1.5×10⁸ colony forming units (CFUs)/mL for the samples to bepulsed. These samples were then plated at dilutions between 10⁻⁶ and 100(no dilution) depending on the effectiveness of the pulse treatments.

Electric Pulse Treatment Protocol. Bacterial samples were then placedinto the 2 mm cuvettes and treated with various EP parameters determinedcomparable to those in the literature (Perni et al. 2007) and selectedsuch that that the highest electric fields induced clinically relevantinactivation (4 log-10) and lower electric fields induced insignificantinactivation (less than 0.3 log-10, or 30%). Although no consensusexists on a universal scaling law for applied EP “dose,” we fixed theenergy densityU=NE² _(τ),   (1)

where N is the number of pulses and τ is the pulse duration. Fixing Ufor E=40, 30, and 20 kV/cm gave N=250, 445, and 1000 pulses,respectively. Cultures were plated as described below.

Cuvettes were filled with 365 μL of sample (sufficient to just cover theelectrodes) and then 250 μL of molecular biology grade mineral oil wasplaced on top. Keeping the samples on ice upon achieving the requireddilution further reduced experimental variability by slowing biologicalprocesses, such as fission. This facilitated the assessment of theinactivation efficiency of EPs, drugs, and combined treatments.

Tests used the clinically relevant, bactericidal drug tobramycin, whichtargets the 30S and 505 ribosome complex and has a clinical dose from 4to 5 μg/mL and rifampicin, which inhibits bacterial dependent RNApolymerase. Added were 0.2, 2, and 20 μg/mL of each drug to the initialbacterial dilutions prior to pulsing. Samples were immediately placed onice to minimize cell division and reduce variability. These aliquotswere also used to plate individual unpulsed controls for each drugconcentration.

Plating. Plating was done on standard disposable tissue culture Petridishes from VWR (15 cm diameter, 10 mm height) and covered with 10 mL ofagar in luria broth (Agar, microbiology tested powder, SIGMA-ALDRICH),which was prepared by adding 20 g of LB lennox (SIGMA-ALDRICH) and 15g/L of agar to water and then autoclaved.

Plated were samples at dilutions of 10⁻⁶ and 10⁻³ for low and highelectric field treatments, respectively, and at 10⁻⁶ and 10⁻⁵ forcontrols. All samples containing 100 μL of pulsed or diluted cultureswere plated on LB agar plates and counted after overnight incubation at37° C.

Pulsing without drugs: Assessed was the impact of EPs on colony formingunits (CFUs) to serve as a baseline for comparing experiments combiningEPs and drugs. Table 1 summarizes the resulting population reduction forboth S. aureus and E. coli for six replicates for the control and 20kV/cm conditions and four replicates for the 30 kV/cm and 40 kV/cmexperiments.

TABLE 1 Electric pulse parameters and the subsequent reduction in S.aureus and E. coli populations. Electric Field # Log reduction Logreduction (kV/cm) Pulses of S. aureus of E. coli 0 0 0.0 ± 0.0 0.0 ± 0.020 1000 0.2 ± 0.1 1.42 ± 0.39 30 445 2.9 ± 0.7 3.26 ± 0.30 40 250 3.7 ±0.7 3.81 ± 0.0 

EPs induced greater cell death for E. coli than for S. aureus until the40 kV/cm. Despite each treatment delivering the same energy density(c.f. Eq. (1)), the applied electric field clearly drove the resultingcell death, as observed previously when assessing the impact of bipolarEP induced ion transport, for each microorganisms with the effectstarting to plateau at 40 kV/cm for E. coli. The rapid increase in CFUreduction observed from 20 kV/cm to 30 kV/cm for each microorganismsuggests a threshold for membrane effects. Electroporation thresholdoccurs for membrane voltages on the order of a few hundred millivoltswith longer durations and higher voltages responsible for making thispermeabilization irreversible.

Thus, simply applying additional lower intensity EPs, even when thesepulse train deposits the same total energy as the higher intensity EPtrain, may be inadequate to inactivate microorganisms. This suggeststhat the time between the pulses (˜1 s) may be sufficiently longcompared to the membrane pore sizes and lifetimes (particularly fornanosecond duration EPs, which typically create smaller pores thanconventional electroporation) for additional EPs to further permeabilizethe membrane. Thus, the pores formed by lower intensity EPs may be toosmall for irreversible pore formation and the time between pulses may besufficiently long so that subsequent pulses do not induce irreversiblepore formation. Thus, subsequent low intensity EPs may be primarilycreating new pores and cells can counter this effect by reestablishinghomeostasis of ions and H+ ions between pulses after the nanoporescollapse.

Pulsing with drugs: bacterial samples of both gram-positive S. aureusand gram-negative E. coli strains were pulsed in a solution with 0.2, 2,and 20 μg/mL of tobramycin and compared to an un-pulsed drug-exposedcontrol. Pulsing and plating were performed to ensure that the drugexposure time of the bacteria was approximately fifteen minutes.

FIG. 1 shows that combining trains of 300 ns EPs with tobramycin inducessignificantly greater inactivation of S. aureus than the antibioticalone. As noted above, even though each train of pulses delivered thesame total energy to the sample, the greatest microorganism inactivationarose for the higher electric fields strengths. Combining the 20 kV/cmEPs with no drug or 0.2 μg/mL induced a 10-20% reduction in S. aureuspopulation. Combining these EP with higher, clinically relevantconcentrations of 2 μg/mL and 20 μg/mL resulted in 1.5 and 2.6log-reduction, respectively, which were statistically significantcompared to treatments of just tobramycin. Although the 30 kV/cm and 40kV/cm conditions resulted in statistically significant inactivationcompared to the untreated control, microorganism inactivation stillincreased with increasing concentration of tobramycin.

FIG. 2 shows that combining trains of 300 ns EPs with 2 and 20 μg/mL ofthe bactericidal tobramycin induces significantly greater inactivationof E. coli than the antibiotic alone. For 0.2 μg/mL tobramycin, the 30and 40 kV/cm treatments exhibited synergy while the 20 kV/cm treatmentdid not. The EPs induce a noticeably greater inactivation of E. colithan S. aureus. The 20 kV/cm condition induced a statisticallysignificant reduction of 1.4 log-10 for the E. coli samples with no drugcompared to sub-log reduction observed for S. aureus. Likewise, the 30kV/cm treatment with no drug induced a 3.5 log-reduction of CFUs, whichis a full log-reduction more compared to the S. aureus. Combining 40kV/cm EPs with antibiotics resulted in reductions of over 9 log-10 andwas considered a complete sterilization of the E. coli. As for S.aureus, increasing the drug concentration enhanced microorganisminactivation.

FIG. 3 shows the impact of rifampicin on S. aureus inactivation.Combining the 20 kV/cm EPs with no drug induced a minimal reduction inS. aureus population. Applying 1/50th of the clinical dose of 10 or 0.2μg/mL, and more clinically relevant concentrations of 2 and 20 μg/mLinduced 1-log reduction. The 30 kV/cm treatment exhibited a greaterclinical significance with the 20 μg/mL dose, causing an additional 1.5log-reduction in 10 min.

FIG. 4 shows the impact of rifampicin on E. coli inactivation. Applying300 ns EPs enhanced the effectiveness of rifampicin in inhibiting E.coli growth. Combining EPs with concentrations of 2 and 20 μg/mL inducedan additional 1.5 log-reduction compared to pulsing alone. Over a 9-logreduction, consistent with complete sterilization, occurred whencombining 20 μg/mL rifampicin with 445 pulses of 30 kV/cm field.Compared to the inactivation due strictly to 20 μg/mL rifampicin with noEPs, the EPs induced a 4-log synergy (additional kill off) compared tojust the rifampicin.

Synergy between antimicrobial composition application and NSEPapplication may be quantified as the additional inactivation induced bythe antimicrobial for a given pulse condition. In addition to thesynergy in the quantity of cell death, the time for inactivation tooccur is also dramatically reduced. While the standard clinical dose of4-5 μg/mL of tobramycin induces a noticeable kill-off of bacteria, thiseffect takes many hours. Combining even 1/20th (0.2 μg/mL) of this dosewith a train of NSEPs causes a 1.5 to 2.5 log-reduction after only 15min of exposure to the antibiotic. FIGS. 5A-5B shows the resultingsynergy for tobramycin for the EP parameters described above. For S.aureus (FIG. 5A), no synergy occurred for the 20 kV/cm pulse trains fortobramycin concentrations at 0.2 μg/mL, while 2.0 μg/mL resulted in 1.5log synergy. For the 30 and 40 kV/cm pulse trains, synergies occurredfor all 2 and 20 μg/mL doses of tobramycin and increased with increasingconcentration. Interestingly, the synergy for tobramycin concentrationsat or above 2 μg/mL was essentially independent of the applied field,although the net inactivation depended strongly on the applied electricfield [c.f. FIG. 1 ]. For E. coli, FIG. 5B shows that pulse trains usingany of the applied electric fields experienced synergy for anytobramycin concentration with the synergy increasing for increasing drugconcentration and relatively insensitive of applied field for both the20 and 30 kV/cm cases. In this case, the synergy appears to decrease forthe 40 kV/cm train when tobramycin concentration is increased from 0.2to 2.0 μg/mL since the total number of viable cells after 40 kV/cmtreatments is less than 2-log from the original 9-log of cells at thebeginning of the experiment.

While one may consider synergy as increasing inactivation byincorporating NSEPs to antibiotic treatment, viewing synergy as a methodof boosting antibiotic effectiveness by reducing time to action hasserious implications in infection treatment. Inducing the effect that anantibiotic would have over a 12 or 24-h period in a matter of seconds tominutes dramatically reduces treatment time, which can be critical forfast-acting infections or infections that are difficult to treat withsuch slow treatment modalities, such as those causing rapid cellulitis.Boosted synergy also implies that lower doses of last-line of defensedrugs, which can have serious side effects, can be used to induce thesame effects as current clinical doses or prophylactic dose levels. Theability of EPs to permeabilize bacterial membranes to improve theeffectiveness of antimicrobials may reduce the number of cells availableto develop resistance.

In these examples, the entire process, including pulsing the sample anddiluting and plating the colonies, took approximately 15 min. Synergyoccurred for 1/20th, 0.2 μg/mL, of the recommended clinical dose oftobramycin, 4 μg/mL, with dramatic synergy arising for higher doses. Thesignificance of this EP-induced synergy is twofold. First, this processwill reduce the antibiotic dose and electrical energy input needed totreat infections and preserve host tissue. Second, the EP may weaken themicroorganisms or sufficiently facilitate passive drug transport intothe cell to allow the application of antibiotics usually ineffectiveagainst certain types or strains of bacteria due to the drug's size orresistance mechanisms present in the bacteria.

Electrochemotherapy, where EPs permeabilize tumors to chemotherapeuticsto enhance treatment efficacy, often use needle electrodes, interlockinggrid patterns of electrodes printed on surfaces, or circular electrodedesigns with a needle electrode surrounded by a cylindrical oppositepolarity electrode. Comparable configurations may be applicable inaccordance with aspects of the present disclosure. Miniaturizedelectrode designs or flexible electronic surfaces (e.g., in the form ofa wand for application at wound sites) may be used as well. Integratingthese electrode designs with drip systems that can deliver drugs orcombination of drugs from vials based on the infection under treatmentis also included.

FIG. 6 shows the synergistic inactivation of Methicillin ResistantStaphylococcus Aureus (MRSA) under 300 ns EPs with combined applicationof tobramycin and rifampicin (same concentration of each drug). FIG. 7shows the effect of 300 ns EPs on MRSA in synergy with different drugs.As shown by conditions without NSEP administration, or withantibacterial dosing that did not show antibacterial effectivenesswithin this time frame, as compared to reductions in CFU when NSEP andantibacterial compositions were applied in combination, the two types oftreatment worked synergistically to produce an antimicrobial effectmeasurably obtained and demonstrated over a short time frame.

Effects on Gram-Negative Bacteria of Gram-Positive Antibacterials inCombination with NSEPS

Methodology was comparable to the foregoing examples. Except forrifampicin, the five other antibiotics in these examples primarilytarget gram-positive bacteria and are largely ineffective or not usedagainst gram-negative bacteria in a clinical setting.

A Blumlein pulsed generator consisting of two lines comprised of twelve2000 pF capacitors connected by inductors produced 300 ns EPs withrise-times of 30 ns and fall-times of 35 ns at a repetition frequency of1 Hz (12). The EPs were applied to a standard 2 mm gap electroporationcuvette (Dot Scientific®) filled with a solution containing themicroorganisms. Used was a Luria broth of 0.5% salinity (5 grams ofNaCl/Liter) for the solution to electrically match the resistance of thesample with the impedance of the pulse generator (11 Ω) to preventsignal reflection. The voltage measurements were taken using a LeCroyPPE 20 kV high voltage probe with a 1000:1 attenuation recorded by aTeleDyne LeCroy® Waverunner 6 Zi Oscilloscope with a bandwidth of 4 GHz.

Sample Preparation:

NSEPs were combined with gram positive antibiotics to enhance theinactivation of three clinically relevant antibiotic resistantgram-negative strains of bacteria and one gram-positive strain. Thegram-negative strains included a carbapenem-resistant Escherichia coli(ATCC® BA-2452™, New Delhi metallo-beta-lactamase NDM-1 positive,),Klebsiella pneumoniae (ATCC® BAA-2146™ NDM-1 positive), and agentamicin, streptomycin and sulfonamide resistant Pseudomonasaeruginosa (BEI Resources NR-31040). Assessed was the gram-positivestrain of methicillin resistant Staphylococcus aureus (USA300-0114strain), which is also resistant to erythromycin and tetracycline.Samples were cultured in Luria broth (LB Broth Lennox, powder microbialgrowth medium, SIGMA-ALDRICH®) by taking 25 mL of broth in a 50 mLsterile conical tube and incubating in a shaker for 16 h at 37° C.Samples were diluted 50% by adding 25 mL media to the incubated sampleas we plated controls for each experiment and condition.

The examples assessed the following antibiotics: vancomycin, linezolid,rifampicin, mupirocin, erythromycin, and fusidic acid. These antibioticsare primarily used against gram-positive bacteria because they cannoteffectively traverse the membranes of gram-negative bacteria. Vancomycinis used primarily to treat gram positive bacteria like MRSA, MRSE, andother resistant strains of enterococci. Linezolid is used primarilyagainst gram-positive bacteria like MRSA, vancomycin resistantenterococci and streptococci. Mupirocin is used primarily to treat MRSAand other S. aureus strains, with mupirocin resistant S. aureus. arisingalmost immediately after clinical trials. Erthromycin is bacteriostaticin nature, and while its mechanism is incompletely understood, it actsinternally by binding to the 50S subunit of the rRNA complex and is alsoused primarily against gram-positive bacteria. Fusidic acid is anotherbacteriostatic compound used primarily to treat gram-positive bacteriawith MRSA and SA strains exhibiting resistance. Of the antibioticsstudied here, the WHO Model list of essential medicines includesrifampicin and vancomycin and classifies linezolid as a drug of lastresort.

Electric Pulse Treatment Protocol:

Samples were treated in 2 mm gap cuvettes containing 365 μL of samplebetween the electrodes, with bio-grade mineral oil added on top of theelectrode plates to prevent arcing. This examples includes starting witha low electric field that induced minimal inactivation alone, but didwhen combined with antibiotics at dosages that were also insufficient toinduce inactivation alone. These results were compared to a higherelectric field capable of inactivating bacteria independently withvarying effectiveness across strains.

To establish a common baseline for comparing different NSEP parameters,we fixed the energy density U delivered to the cuvettes according toFormula (1) (above). For 300 ns EPs, 500 EPs at 20 kV/cm and 222 EPs at30 kV/cm delivered the same energy to the sample.

Plating:

Tissue culture Petri dishes from VWR® (15 cm diameter, 10 mm height)were utilized for plating. Each plate was covered with 15 mL of Luriabroth (Agar, microbiology tested powder, SIGMA-ALDRICH®), which wasprepared by adding 20 g of LB Lennox (SIGMA-ALDRICH) and 15 g/L of agarto water prior to being autoclaved. The salinity of the agar was thesame as the LB (0.5% NaCl) to minimize additional environmentalstressors on the bacteria.

Removed was 20 μL from each cuvette and added to 180 μL of PBS in a96-well dish in triplicate. Each well was then diluted by a ratio of10:1 five additional times by taking 20 μL of the diluted sample intosubsequent wells using multi-channel pipettes (using fresh tips for eachdilution). Plated were the six dilutions of each sample two times eachby adding 4 μL from each well onto the Petri dish using a multi-channelpipette, which allowed the plating of all three replicates on the sameplate to achieve n=6. These plates were then cultured overnight in anincubator at 37° C. and counted the next day.

Counts were taken for relevant dilutions (colony counts ranged from 15to 25 colonies) and multiplied by 25×10^(dilution), to account for the 4μL plated for each condition. Examples were repeated three times eachover different days with different incubated samples. Except forrifampicin, the five other antibiotics in these examples primarilytarget gram-positive bacteria and are largely ineffective or not usedagainst gram-negative bacteria in a clinical setting.

Bacterial inactivation combining Electric Pulses (EPs) with antibiotics

Assessed were various combinations of the antibiotics above with NSEPsfor inactivating one gram-positive and three-gram negative bacteria.FIGS. 8A-8B highlight the inactivation of MRSA 300 under theseconditions and the resulting synergy that arises from adding a singleantibiotic to the NSEPs. Treating MRSA 300 with either 2 μg/mL or 20μg/mL induced less than 1-log reduction in cell count. Applying either445 20 kV/cm or 222 30 kV/cm 300 ns EPs induced over 1-log or 4-logreduction, respectively. Since these NSEPS had the same pulse durationand delivered the same overall energy density to the microbes, and thetime between EPs of 1 s may be of sufficient for many pores to reseal,this suggests that higher electric field may have a dramatic effect onMRSA 300 inactivation. With all else equal, raising the electric fieldmay increase membrane potential, making both reversible and irreversibleelectroporation more likely. Because only very long-lived pores willremain for either NSEP condition, the driving factor for membrane leveleffects will likely be the membrane potential from a single NSEP,suggesting that the 30 kV/cm should have the larger impact. FIG. 8Afurther shows that, for example, combinations of 2 μg/mL of rifampicinwith the 20 kV/cm NSEPs and 20 μg/mL of mupirocin or rifampicin with the30 kV/cm NSEPs yielded improvement on MRSA 300 inactivation compared tousing only the NSEPs.

FIG. 8B shows the synergistic inactivation caused by adding antibioticsto the NSEPs. As anticipated from FIG. 8A, the largest synergy ofapproximately 1.5-log occurred when combining 2 μg/mL of rifampicin withthe 20 kV/cm NSEPs and 20 μg/mL rifampicin and mupirocin with the 30kV/cm EPs. The increased synergy of mupirocin with increasing electricfield suggests that permeabilizing the membrane to facilitate itsconcentration in the MRSA may be particularly important for improvedtreatment outcomes since it is specifically designed to target MRSA.

Inactivating Gram-Negative Bacteria with Gram Positive Antibiotics

FIGS. 9A-9B show inactivation for the NSEPs and/or either 2 μg/mL or 20μg/mL of linezolid, mupirocin, rifampicin, or vancomycin. 20 kV/cm NSEPsinduce less than 1-log reduction in the gram-negative K. pneumoniae,while the 30 kV/cm NSEPs induce a statistically significant 3.5-logreduction. Applying 2 μg/mL or 20 μg/mL of the four drugs induced nostatistically significant reduction in K. pneumoniae. Combining any ofthe drugs with the 20 kV/cm NSEPs induced a slight increase 2 μg/mLmupirocin and an increase for the 20 μg/mL mupirocin. For the 30 kV/cmNSEPs, adding 2 μg/mL of rifampicin or vancomycin induced 5-logreduction and 2 μg/mL induced a 4-log inactivation. Adding 20 μg/mL ofmupirocin or vancomycin to the 30 kV/cm NSEPs induced over 4-logreduction that was an improvement over the NSEPs. Adding 20 μg/mL ofeither linezolid or rifampicin did not induce a statisticallysignificant improvement over the NSEPs themselves.

FIG. 9B quantifies improvement by looking at the synergistic(additional) inactivation induced by the drugs compared to just theNSEPs alone. Combining the drugs with the 20 kV/cm EPs induced noadditional inactivation. Combining 2 μg/mL of mupirocin, rifampicin, orvancomycin to the 30 kV/cm NSEPs induced 1-log additional inactivationthat was a statistical significant improvement. Adding 20 μg/mL ofmupirocin and vancomycin to the 30 kV/cm NSEPs induced over 1-logadditional inactivation; adding 20 μg/mL of rifampicin induced 1-logadditional inactivation. Thus, these results indicate that NSEPs caneffectively make antibiotics that normally target gram-negativebacterial (mupirocin and vancomycin) more effective against agram-negative bacterium than either the NSEPs or the drug alone.

FIGS. 10A-10B show similar inactivation of gram-negative E. coli by grampositive antibiotics. Applying 2 μg/mL or 20 μg/mL of linezolid, fusidicacid, erythromycin, mupirocin, firampicin, or vancomycin induced changesin E. coli population. Applying the 20 kV/cm NSEPs induced a 1-loginactivation. Combining the 20 kV/cm NSEPs with these 2 μg/mLantibiotics inactivated between 2-log and 3-log E. coli compared to theNSEPs alone. Adding 20 μg/mL of these antibiotics induced similar levelsof inactivation. The consistent inactivation levels when combining the20 kV/cm NSEPs with the either 2 μg/mL and 20 μg/mL suggests that the 20kV/cm NSEPs are sufficient to enhance the synergistic benefit with theantibiotics at 2 μg/mL, meaning that additional levels of the drugs donot provide benefit for the timescales assessed here. Applying the 30kV/cm NSEPs induced 2-log reduction. Combining the 30 kV/cm NSEPs with 2μg/mL of these antibiotics resulted in a statistically significantimprovement to 3.5 to 5-log reduction and combining with 20 μg/mLresulted in 3.5 to 6-log reduction with the 6-log reduction involvingthe combination with rifampicin. FIG. 10B shows that 20 kV/cm NSEPsinduce synergy for all the 2 μg/mL drugs except for vancomycin and forthe 20 μg/mL only for linezolid and rifampicin. For the 30 kV/cm NSEPs,the drugs induced from between 1.5- to 4-log synergy. Thus, NSEPs againinduce the gram-negative drugs to effectively inactivate a gram-positivebacterium.

For P. aeruginosa, a biofilm forming bacteria, FIGS. 11A-11B show thatthe drugs alone induced no activation, the 20 kV/cm NSEPs inducedapproximately 1-log reduction, and the 30 kV/cm NSEPs induced less than1-log reduction. In this case, combining 2 μg/mL or 20 μg/mL oflinezolid, erythromycin, mupirocin, rifampicin, or vancomycin with the20 kV/cm NSEPs induced no additional inactivation (and in several casesactually induced less inactivation). For the 30 kV/cm NSEPs, adding 2μg/mL induced up to 2-log reduction for vancomycin. Combining 20 μg/mLwith the 30 kV/cm NSEPs caused improvements in inactivation except forvan.

FIG. 11B shows that combining 2 μg/mL of vancomycin leads to synergywith 30 kV/cm NSEPs. Results demonstrate an improvement when applying 30kV/cm NSEPs compared to the 20 kV/cm NSEPs.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the present disclosure andthese are therefore considered to be within the scope of the presentdisclosure as defined in the claims that follow.

What is claimed is:
 1. A method of treating a localized bacterialinfection in an animal, said method comprising: contacting microbes withan antibiotic compound and applying pulses of electricity having aduration of between about 50 nanoseconds and about 900 nanoseconds,wherein the applying pulses of electricity are to a surface of theanimal, and wherein the surface comprises the localized infection. 2.The method of claim 1, wherein the pulses of electricity have anintensity and the intensity is between about 20 kV/cm and about 40kV/cm.
 3. The method of claim 1, wherein the pulses of electricity havean intensity and the intensity is between about 1 kV/cm and about 20kV/cm.
 4. The method of claim 1, wherein the pulses of electricity areapplied at a frequency of between about 0.1 Hz and about 10 Hz.
 5. Themethod of claim 1, wherein said microbes are a strain of bacteria andthe antibiotic is applied at a concentration for a duration, whereinapplying the antibiotic to the strain at the concentration for theduration does not reduce a viable number of bacteria of the strain whenthe pulses of electricity are not also applied to the strain.
 6. Themethod of claim 1, wherein the microbes comprise a gram-negative strainof bacteria.
 7. The method of claim 1, wherein the microbes comprise agram-positive strain of bacteria.
 8. The method of claim 1, wherein saidmicrobes are a strain of bacteria and the antibiotic is applied at aconcentration for a duration, wherein the treating or preventing isgreater when the antibiotic is applied to the strain at theconcentration for the duration when the pulses of electricity areapplied to the strain than when the antibiotic is applied to the strainat the concentration for the duration when the pulses of electricity arenot applied to the strain.
 9. The method of claim 8, wherein themicrobes comprise a gram-negative strain of bacteria.
 10. The method ofclaim 8, wherein the microbes comprise a gram-positive strain ofbacteria.
 11. The method of claim 1, wherein the antibiotic is selectedfrom at least one of an aminoglycoside antibiotic, an ansamycinantibiotic, a beta-lactam antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a lipopeptide antibiotic, a macrolideantibiotic, a monobactam antibiotic, a nitrofuran antibiotic, anoxazolidinone antibiotic, a quinolone antibiotic, a fluoroquinoloneantibiotic, a sulfonamide antibiotic, a tetracycline antibiotic,pexiganan, fusidic acid, mupirocin, and any combination of at least twoof the foregoing.
 12. The method of claim 1, wherein the antibiotic isselected from at least one of tobramycin, streptomycin, rifampicin,vancomycin, clindamycin, daptomycin, erythromycin, linezolid,penicillin, minocycline, pexiganan, fusidic acid, mupirocin, bacitracin,neomycin, polymixin B, metronidazole, silver, zinc, copper, and anycombination of at least two of the foregoing.
 13. The method of claim 1,wherein the microbe is Staphylococcus aureus, Staphylococcusepidermidis, Escherichia coli, Acenitobacter baumanii, Klebsiellapneumoniae, or Pseudomonas aeruginosa.
 14. The method of claim 1,wherein the microbe is vancomycin-resistant Staphylococcus aureus,methicillin-resistant Staphylococcus aureus, a strain ofmultidrug-resistant Pseudomonas aeruginosa, or a strain ofmultidrug-resistant Escherichia coli.
 15. The method of claim 1, whereinthe animal is a human, wherein contacting further comprisesadministering the antibiotic to a human, and applying further comprisesapplying the electric pulses to the human.
 16. The method of claim 15,wherein administering is selected from administering topically andadministering systemically.
 17. The method of claim 1, wherein thelocalized infection is external or internal to the animal.
 18. Themethod of claim 1, further comprising applying the pulses of electricityto a surface of an implanted object, wherein the surface of theimplanted object comprises the localized infection.